Rats (Rattus norvegicus) are a widely used animal model in the fields of hypertension, cardiovascular physiology, diabetes, metabolic disorders, behavioral studies and toxicity testing. Michalkiewicz et al. (2007) J. Amer. Phys. Society 293:H881-H894. The availability of these model systems, advances in rat genomics and sequence of the rat, human and mouse genomes have greatly accelerated the use of inbred rat models for discovery of the genetic basis of complex diseases and provided animal models for therapeutic drug discovery.
However, the advances in the information about the rat genome have not been accompanied by parallel progress in genome modification technology. Unlike mice, rat embryonic stem cell clones for gene targeting are not readily produced. Pronuclear injection has also proven difficult and has a poor success rate in generating transgenic rats. Michalkiewicz et al. (2007) J. Amer. Phys. Society 293:H881-H894 report generation of transgenic rats using a lentiviral construct expressing an enhanced green fluorescent protein (eGFP) reporter gene, where the eGFP transgene was found to be present in 1-4 copies integrated at random sites within the genome.
There remains a need for methods of modifying rat genomes in a targeted fashion. Precisely targeted site-specific cleavage of genomic loci offers an efficient supplement and/or alternative to conventional homologous recombination. Creation of a double-strand break (DSB) increases the frequency of homologous recombination at the targeted locus more than 1000-fold. More simply, the imprecise repair of a site-specific DSB by non-homologous end joining (NHEJ) can also result in gene disruption. Creation of two such DSBs results in deletion of arbitrarily large regions. The modular DNA recognition preferences of zinc-fingers protein allows for the rational design of site-specific multi-finger DNA binding proteins. Fusion of the nuclease domain from the Type II restriction enzyme Fok I to site-specific zinc-finger proteins allows for the creation of site-specific nucleases. See, for example, United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20060063231; 20070134796; 2008015164; 20080131962; 2008015996 and International Publication WOs 07/014,275 and 2008/133938, which all describe use of zinc-finger nucleases and which are incorporated by reference in their entireties for all purposes.